Isolation and enumeration of Campylobacter jejuni from poultry products by a selective enrichment method

A direct selective enrichment procedure was developed for the isolation of Campylobacter jejuni from poultry products. The selective enrichment medium (ATB) consisted of (per liter) tryptose (20 g), yeast extract (2.5 g), sodium chloride (5 g), FBP supplement (ferrous sulfate [0.25 g], sodium metabisulfite [0.25 g], sodium pyruvate [0.25 g]), bicine (10 g), and agar (1 g). Hematin solution (6.25 ml; prepared by dissolving 0.032 g of bovine hemin in 10 ml of 0.15 N sodium hydroxide solution and autoclaving at 0.35 kg/cm2 for 30 min), rifampin (25 mg), cefsulodin (6.25 mg), and polymyxin B sulfate (20,000 IU) were added after the medium was sterilized. The pH was adjusted to 8.0. Samples were enriched in the above medium at 42 degrees C for 48 h under an atmosphere of 5% O2, 10% CO2, and 85% N2. Enrichment cultures were streaked on a plating medium composed of Brucella agar, hematin solution, FBP supplement, and the above antibiotics. Plates were incubated under the same conditions as above. Suspect colonies from the plates were confirmed to be C. jejuni by morphological examination, growth characteristics, and biochemical tests. The above method yielded 25 isolates of C. jejuni from 50 samples of retail cut-up chicken and chicken parts, whereas a more complex method involving filtration, centrifugation, selective enrichment under a flowing atmosphere, and membrane filtration yielded only 6 positives from the same samples. The new isolation procedure was particularly effective in isolating C. jejuni in the presence of large numbers of Pseudomonas aeruginosa.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selected enrichment broths for recovery of Campylobacter jejuni from foods.

We attempted to shorten the required time for enrichment broth culture for the isolation of Campylobacter jejuni. Enrichment broths described by Doyle and Roman and Park and Stankiewicz and one developed during this study were compared for ability to isolate C. jejuni from raw chicken carcasses. Our medium was a modification of that of Doyle and Roman with the addition of filter-sterilized FBP (0.2% ferrous sulfate, 0.025% sodium metabisulfite, 0.05% sodium pyruvate), 0.1% sodium lauryl sulfate, and 0.075% agar. Initially, laboratory strains were employed in the development of this medium. Subsequently, an indigenous load of C. jejuni obtained from chickens was used to compare media. Isolation rate comparisons were as follows: direct plating, 40%; Doyle and Roman broth, 45% at 7 h and 61% at 16 h; Park and Stankiewicz broth, 53% at 7 h and 60% at 16 h; our broth, 48% at 7 h and 50% at 16 h. In addition to having the highest isolation rate, the enrichment broth of Doyle and Roman showed greatest selectivity. Our inoculation method of indigenous bacteria provided a controlled means for comparison of isolation procedures.     

Efficacy of media and methods for detecting and enumerating Campylobacter jejuni in refrigerated chicken meat.

A study was undertaken to compare several enrichment and direct isolation media for their suitability to detect and enumerate five strains of Campylobacter jejuni in refrigerated (5 degrees C) chicken meat. The influence of CO2 on survival at 5 degrees C was also investigated. Selective enrichment media evaluated included Preston broth (PB), selective semisolid brucella medium (SSBM), Campylobacter enrichment broth (CEB), VTP brucella-FBP broth (VTP), Rosef and Kapperud Campylobacter enrichment broth (RKCEB), and Doyle and Roman enrichment broth (DREB). Direct isolation agars included Campy brucella agar (CBAP), blood-free Campylobacter medium (BFCM) and modified Butzler agar (MBA). Comminuted chicken meat was inoculated with C. jejuni, sealed under atmospheric gas or CO2, and stored at 5 degrees C for up to 21 days. Viable population was determined by the most-probable-number technique (PB, SSBM, CEB, VTP, and RKCEB, followed by plating on CBAP, BFCM, and MBA), enrichment on DREB, followed by plating on CBAP, BFCM, and MBA, and direct isolation on CBAP, BFCM, and MBA. Without exception, direct plating of samples was superior to the most-probable-number technique for enumerating C. jejuni; MBA was inferior to CBAP and BFCM, and DREB performed at least as well as other enrichment media evaluated. Carbon dioxide afforded protection against death of three of the five strains of C. jejuni tested.     

Efficacy of media and methods for detecting and enumerating Campylobacter jejuni in refrigerated chicken meat

A study was undertaken to compare several enrichment and direct isolation media for their suitability to detect and enumerate five strains of Campylobacter jejuni in refrigerated (5 degrees C) chicken meat. The influence of CO2 on survival at 5 degrees C was also investigated. Selective enrichment media evaluated included Preston broth (PB), selective semisolid brucella medium (SSBM), Campylobacter enrichment broth (CEB), VTP brucella-FBP broth (VTP), Rosef and Kapperud Campylobacter enrichment broth (RKCEB), and Doyle and Roman enrichment broth (DREB). Direct isolation agars included Campy brucella agar (CBAP), blood-free Campylobacter medium (BFCM) and modified Butzler agar (MBA). Comminuted chicken meat was inoculated with C. jejuni, sealed under atmospheric gas or CO2, and stored at 5 degrees C for up to 21 days. Viable population was determined by the most-probable-number technique (PB, SSBM, CEB, VTP, and RKCEB, followed by plating on CBAP, BFCM, and MBA), enrichment on DREB, followed by plating on CBAP, BFCM, and MBA, and direct isolation on CBAP, BFCM, and MBA. Without exception, direct plating of samples was superior to the most-probable-number technique for enumerating C. jejuni; MBA was inferior to CBAP and BFCM, and DREB performed at least as well as other enrichment media evaluated. Carbon dioxide afforded protection against death of three of the five strains of C. jejuni tested.     

Comparison of some enrichment broths and growth media for the isolation of thermophilic campylobacters from surface water samples

Three different enrichment broths and two selective growth media were compared for isolating thermophilic campylobacters by combined membrane filtration and enrichment techniques from surface waters of different physical, chemical and bacteriological characteristics. Fifty-two strains of campylobacters were isolated from total of 1668 cultures. The various broth/medium combinations did not affect the dominance of C. jejuni over C. coli (total 49 C. jejuni and three C. coli). The most efficient combinations of enrichment broth and growth media were either Oosterom broth/blood-free charcoal-cefoperazone-deoxycholate agar (CCDA) medium or blood-free charcoal-cefoperazone-deoxycholate (CCD) broth/CCDA medium. Modified Preston broth (sheep blood instead of horse blood) with either of the growth media gave significantly lower yields although it suppressed efficiently the growth of contaminants. Skirrow medium had lower selectivity than CCDA medium and gave slightly lower isolation rate. Enrichment time (24 or 48 h) did not affect the isolation frequency of campylobacters but longer enrichment time increased the growth of contaminants. Prefiltration through membranes of pore sizes 5.0 and 1.2 microns decreased the growth of contaminants. However, these membranes retain campylobacters and must be cultured to avoid underestimation. From more polluted waters campylobacters were isolated most frequently with CCD broth and CCDA medium.     

Survival of Campylobacter jejuni strains of different origin in drinking water

AIMS: The aim of the study was to measure the survival of 19 Campylobacter jejuni strains of different origins, including two reference strains, four poultry-derived isolates, nine human isolates and four water isolates, in sterilized drinking water. METHODS AND RESULTS: Pure cultures of 19 C. jejuni strains were inoculated in sterile drinking water and incubated at 4 degrees C for 64 days. Survival was determined by culturability on both selective (Karmali agar) and non-selective [Columbia blood agar (CBA)] media. Culturability was shown to be strain and origin-dependent. Campylobacter jejuni showed prolonged survival on a non-selective than on a selective medium. CONCLUSIONS: The origin of the strain is a determining factor for the survival of C. jejuni in drinking water at 4 degrees C. Poultry isolates showed a prolonged survival, which could be an indication that these strains could play an important role in the transmission of campylobacteriosis through water. In addition, culture conditions are an important factor for evaluating the survival of C. jejuni in drinking water at 4 degrees C. The non-selective agar (CBA) allowed growth of C. jejuni over a longer period of time than the selective agar (Karmali). Furthermore, an enrichment broth (Bolton) allowed the recovery of all 19 C. jejuni strains during the 64 days of incubation at 4 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlighted differences in culturability depending on culture conditions and on strain origin.     

Chicken juice, a food-based model system suitable to study survival of Campylobacter jejuni

AIMS: The purpose of this study was to develop a food-based model system that resembles the environment that Campylobacter jejuni experiences on raw poultry products and use this model system to investigate growth and survival of the bacterium. METHODS AND RESULTS: Chicken juice was collected from frozen chickens and subsequently cleared by centrifugation and subjected to sterile filtration. At low temperatures (5 and 10 degrees C) C. jejuni NCTC11168 remained viable in chicken juice for a remarkably longer period of time than in the reference medium BHI. When exposed to heat stress (48 degrees C) C. jejuni NCTC11168 also showed increased viability in chicken juice compared with the reference medium. Furthermore, agar plates made with chicken juice supported growth of four clinical isolates of C. jejuni and a C. jejuni strain obtained from chicken at both 37 and 42 degrees C. CONCLUSIONS: Our work shows that minimal processed and sterilized chicken juice is an ideal environment for survival of C. jejuni and that it is useful as a food-based model system. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed model system may contribute to the understanding of C. jejuni viability on poultry products and can be instrumental in the development of alternative preservation strategies.     

Detection and genotyping of Arcobacter and Campylobacter isolates from retail chicken samples by use of DNA oligonucleotide arrays

To explore the use of DNA microarrays for pathogen detection in food, we produced DNA oligonucleotide arrays to simultaneously determine the presence of Arcobacter and the presence of Campylobacter in retail chicken samples. Probes were selected that target housekeeping and virulence-associated genes in both Arcobacter butzleri and thermotolerant Campylobacter jejuni and Campylobacter coli. These microarrays showed a high level of probe specificity; the signal intensities detected for A. butzleri, C. coli, or C. jejuni probes were at least 10-fold higher than the background levels. Specific identification of A. butzleri, C. coli, and C. jejuni was achieved without the need for a PCR amplification step. By adapting an isolation method that employed membrane filtration and selective media, C. jejuni isolates were recovered from package liquid from whole chicken carcasses prior to enrichment. Increasing the time of enrichment resulted in the isolation of A. butzleri and increased the recovery of C. jejuni. C. jejuni isolates were further classified by using an additional subset of probes targeting the lipooligosaccharide (LOS) biosynthesis locus. Our results demonstrated that most of the C. jejuni isolates likely possess class B, C, or H LOS. Validation experiments demonstrated that the DNA microarray had a detection sensitivity threshold of approximately 10,000 C. jejuni cells. Interestingly, the use of C. jejuni sequence-specific primers to label genomic DNA improved the sensitivity of this DNA microarray for detection of C. jejuni in whole chicken carcass samples. C. jejuni was efficiently detected directly both in package liquid from whole chicken carcasses and in enrichment broths.     

Recovery of Campylobacter jejuni and Campylobacter coli from inoculated foods by selective enrichment.

A direct enrichment procedure was developed to selectively recover small numbers of Campylobacter jejuni, C. coli, and nalidixic acid-resistant thermophilic Campylobacter from foods. The procedure includes an enrichment medium composed of brucella broth, 7% lysed horse blood, 0.3% sodium succinate, 0.01% cysteine hydrochloride, vancomycin (15 micrograms/ml), trimethoprim (5 micrograms/ml), polymyxin B (20 IU/ml), and cycloheximide (50 micrograms/ml) that is inoculated with 10 or 25 g of food and incubated with agitation under microaerophilic conditions at 42 degrees C for 16 to 18 h. After incubation, the medium is plated directly onto Campy-BAP agar plates (M. J. Blaser et al., Ann. Intern. Med. 91:179-185, 1979), and resulting colonies that resemble Campylobacter are identified by conventional tests. The foods evaluated included raw milk, hamburger, and chicken skin which had aerobic plate counts of 10(5) to 10(9) bacteria/g. The procedure was effective in recovering as few as 0.1 cell of Campylobacter per g of food. Of the 50 isolates of Campylobacter evaluated, all were recovered from raw milk and hamburger at a level of 1 to 4 cells/g, and 41 and 40 isolaes were recovered from the hamburger and milk, respectively, at 0.1 to 0.4 cell/g. The enrichment was least effective for recovering campylobacters from chicken skin, as 7 and 26 of 50 isolates were not recovered at 1 to 4 and 0.1 to 0.4 cell/g, respectively. This new procedure is more rapid, direct, and effective than other enrichment or direct plating procedures for recovering small numbers of campylobacters from foods.     

Detection of Campylobacter jejuni added to foods by using a combined selective enrichment and nucleic acid sequence-based amplification (NASBA).

An assay to detect Campylobacter jejuni in foods that uses a short selective enrichment culture, a simple and rapid isolation procedure, NASBA amplification of RNA, and a nonradioactive in solution hybridization was studied. The presence of high numbers of indigenous flora affected the sensitivity of the assay. However, detection of C. jejuni was possible up to a ratio of indigenous flora to C. jejuni of 10,000:1. Interference by food components was eliminated by centrifugation following the enrichment step. Fourteen food samples artificially inoculated with C. jejuni (1 to 1,000 CFU/10 g) were analyzed with the NASBA assay and the conventional culture method with Campylobacter charcoal differential agar (CCDA). A few false-negative results were obtained by both NASBA (1.42%) and CCDA (2.86%) isolation. Yet the use of enrichment culture and NASBA shortened the analysis time from 6 days to 26 h. The relative simplicity and rapidity of the NASBA assay make it an attractive alternative for detection of C. jejuni in food samples.     

Methods for isolating Campylobacter jejuni from low-turbidity water

Membrane filtration methods were developed and evaluated for the quantitative recovery of Campylobacter jejuni from environmental waters of low turbidity. The best procedure studied involved passaging the test water through a filter (pore size, 0.45 micron) and plating it facedown on Campylobacter-selective agar. The filter was removed after overnight incubation, and the plate was streaked for isolation and then reincubated. This method, with or without prefiltration through 5.0- and 0.6-micron-pore-size membranes consistently resulted in the recovery of 30 C. jejuni CFU/250 ml of seeded natural waters. The other methods, plating the final filter face-up or preincubation of the filter in an enrichment medium, were not as sensitive. The technique described above could be useful in the routine monitoring of finished waters for C. jejuni or during investigations of suspected waterborne outbreaks for water of low turbidity.     

Methods for isolating Campylobacter jejuni from low-turbidity water.

Membrane filtration methods were developed and evaluated for the quantitative recovery of Campylobacter jejuni from environmental waters of low turbidity. The best procedure studied involved passaging the test water through a filter (pore size, 0.45 micron) and plating it facedown on Campylobacter-selective agar. The filter was removed after overnight incubation, and the plate was streaked for isolation and then reincubated. This method, with or without prefiltration through 5.0- and 0.6-micron-pore-size membranes consistently resulted in the recovery of 30 C. jejuni CFU/250 ml of seeded natural waters. The other methods, plating the final filter face-up or preincubation of the filter in an enrichment medium, were not as sensitive. The technique described above could be useful in the routine monitoring of finished waters for C. jejuni or during investigations of suspected waterborne outbreaks for water of low turbidity.     

A real-time PCR assay for the detection of Campylobacter jejuni in foods after enrichment culture

A real-time PCR assay was developed for the quantitative detection of Campylobacter jejuni in foods after enrichment culture. The specificity of the assay for C. jejuni was demonstrated with a diverse range of Campylobacter species, related organisms, and unrelated genera. The assay had a linear range of quantification over six orders of magnitude, and the limit of detection was approximately 12 genome equivalents. The assay was used to detect C. jejuni in both naturally and artificially contaminated food samples. Ninety-seven foods, including raw poultry meat, offal, raw shellfish, and milk samples, were enriched in blood-free Campylobacter enrichment broth at 37 degrees C for 24 h, followed by 42 degrees C for 24 h. Enrichment cultures were subcultured to Campylobacter charcoal-cefoperazone-deoxycholate blood-free selective agar, and presumptive Campylobacter isolates were identified with phenotypic methods. DNA was extracted from enrichment cultures with a rapid lysis method and used as the template in the real-time PCR assay. A total of 66 samples were positive for C. jejuni by either method, with 57 samples positive for C. jejuni by subculture to selective agar medium and 63 samples positive in the real-time PCR assay. The results of both methods were concordant for 84 of the samples. The total time taken for detection from enrichment broth samples was approximately 3 h for the real-time PCR assay, with the results being available immediately at the end of PCR cycling, compared to 48 h for subculture to selective agar. This assay significantly reduces the total time taken for the detection of C. jejuni in foods and is an important model for other food-borne pathogens.     

Pre-Enrichment and Enrichment Methods for Isolating Small Number of Campylobacteria from Contaminating Flora

A method was developed to detect fewer than 100 CFU of campylobacteria from SIFF transport medium to which thawing drip from deep frozen broiler carcasses was added as a source of contamination and which was then stored at room temperature for 20 h. The method was made possible by using pre–enrichment in 1 % buffered peptone water under a microaerophilic atmosphere for 5 h at 43°C before selective enrichment either in brucella enrichment broth and on brucella blood selective agar supplemented with Skirrow antibiotics or in CCD enrichment broth and on blood free CCD selective agar. The other pre–enrichment broth studied was alkaline peptone water with reducing agents (RAPW) and the other enrichment broths and selective agars were Preston broth and agar, THAL broth and alkaline tryptose broth (ATB) and brucella agar with ATB antibiotics. Contaminating flora can be a problem when using enrichment broths and selective agars with limited antibiotic supplementation.     

Prevalence of campylobacters and arcobacters in ducks at the abattoir

Ten duck carcasses, five from each of two different flocks, and four pairs of pooled duck caecal contents, each pair from a separate flock, were examined by a variety of techniques for arcobacters and campylobacters. Campylobacter coli, C. jejuni ssp. jejuni , C. upsaliensis, Arcobacter cryaerophilus and A. butzleri were isolated from duck caecal contents. Campylobacter coli, C. jejuni ssp. jejuni, A. cryaerophilus, A. butzleri and A. skirrowii were isolated from carcasses. The most effective methods for isolating these bacteria from carcasses involved selective enrichment in campylobacter enrichment broth, containing a cefoperazone, amphotericin, teicoplanin supplement, followed by plating onto modified charcoal cefoperazone deoxycholate agar (mCCDA), or plating onto non-selective blood agar after filtration through a 0·65 μm pore size cellulose acetate filter. In contrast, recovery from caecal contents was most effective by direct plating onto mCCDA. API test strips performed poorly, failing to identify A. skirrowii or A. butzleri (which are not included in the scheme), or even many common campylobacters. The Preston biochemical characterization scheme was more helpful, though it did not distinguish between Arcobacter species. The species of most isolates of campylobacter, identified using the Preston scheme, was confirmed by the use of SDS-PAGE of whole cell proteins and this technique was also used successfully to speciate arcobacters.     

Selective and Differential Media for Isolation and Tentative Identification of Each Species of Pityrosporum Residing on Normal or Diseased Human Skin

Selective and differential media were designed for each species of Pityrosporum; P. pachydermatis, P. ovale, and P. orbiculare in order to make feasible a quantitative cultivation. Medium for P. pachydermatis (medium A) was composed of 1% trypticase peptone (BBL), 0.5% yeast extract (BBL), 0.3% glucose, 0.2% NaCl, 1.2% KH₂ PO₄ (anhydrous), 1.5% agar, 0.01% ampicillin, and 0.025% cycloheximide with a pH of 5.5. Medium for P. ovale (medium B) was medium A supplemented with 0.05% sodium acetate (anhydrous), 0.2% Tween 80, and 0.025% (selective medium) or 0.075% (differential medium) sodium laurate. Medium for P. orbiculare was medium B (devoid of laurate) supplemented with 2% olive oil, 0.25% glycerol, 0.25% gall powder, 0.05% sodium palmitate, 0.05% sodium stearate, 0.05% sodium oleate and 8% (selective medium) or 10% (differential medium) sodium lactate and an increase in Tween to 1%. For isolation of Pityrosporum, specimens were suspended in 0.1% Tween 80 solution and inoculated onto agar plates of three selective media. The plates were incubated aerobically at 37 C for 8–10 days under conditions of prevention of water loss from the media. The plating efficiency of each selective medium, expressed as a ratio of cultural counts to microscopic counts was generally over 70%. Species of Pityrosporum could also be identified when we inoculated the cell suspension onto differential agar plates and incubated the preparations at 37 C for 7 days.     

Survival of cold-stressed Campylobacter jejuni on ground chicken and chicken skin during frozen storage

Campylobacter jejuni is prevalent in poultry, but the effect of combined refrigerated and frozen storage on its survival, conditions relevant to poultry processing and storage, has not been evaluated. Therefore, the effects of refrigeration at 4 degrees C, freezing at -20 degrees C, and a combination of refrigeration and freezing on the survival of C. jejuni in ground chicken and on chicken skin were examined. Samples were enumerated using tryptic soy agar containing sheep's blood and modified cefoperazone charcoal deoxycholate agar. Refrigerated storage alone for 3 to 7 days produced a reduction in cell counts of 0.34 to 0.81 log10 CFU/g in ground chicken and a reduction in cell counts of 0.31 to 0.63 log10 CFU/g on chicken skin. Declines were comparable for each sample type using either plating medium. Frozen storage, alone and with prerefrigeration, produced a reduction in cell counts of 0.56 to 1.57 log10 CFU/g in ground chicken and a reduction in cell counts of 1.38 to 3.39 log10 CFU/g on chicken skin over a 2-week period. The recovery of C. jejuni following freezing was similar on both plating media. The survival following frozen storage was greater in ground chicken than on chicken skin with or without prerefrigeration. Cell counts after freezing were lower on chicken skin samples that had been prerefrigerated for 7 days than in those that had been prerefrigerated for 0, 1, or 3 days. This was not observed for ground chicken samples, possibly due to their composition. C. jejuni survived storage at 4 and -20 degrees C with either sample type. This study indicates that, individually or in combination, refrigeration and freezing are not a substitute for safe handling and proper cooking of poultry.     

Growth of Desulfovibrio on the Surface of Agar Media

Growth of Desulfovibrio desulfuricans (API strain) was found to take place in an atmosphere of hydrogen on the agar surface of complex media, including yeast extract (Difco), and Trypticase Soy Agar (BBL) without any added reducing agents. For growth on a 2% yeast extract-agar surface in the absence of hydrogen (nitrogen atmosphere), sodium lactate was required in the medium. Growth on the surface of Trypticase Soy Agar (TSA) under nitrogen took place readily in the absence of an added hydrogen donor. A medium (TSA plus salts) is described based upon the addition of sodium lactate (4 ml per liter), magnesium sulfate (2 g per liter), and ferrous ammonium sulfate (0.05%) to TSA, which appears suitable for the isolation and growth of Desulfovibrio on the surface of agar plates in an atmosphere of hydrogen. Sodium lactate does not appear to be essential in this medium for good growth and sulfate reduction in a hydrogen atmosphere, but is essential in a nitrogen atmosphere. Growth of Desulfovibrio (hydrogen atmosphere) on the agar surface of media commonly used for its cultivation as well as on an inorganic medium containing bicarbonate as a source of carbon is poor and erratic unless inoculated (Desulfovibrio) plates of TSA plus salts are incubated in the same container with plates of these media. This stimulatory effect of incubation with inoculated plates of TSA plus salts medium appears to be due to as yet unidentified volatile material produced by D. desulfuricans when growing on this medium. Another volatile material, or possibly the identical material, appears to act similarly to a hydrogen donor.     

Campylobacter jejuni survival in chicken meat as a function of temperature.

Recognition of Campylobacter fetus subsp. jejuni (referred to hereafter as C. jejuni) as an important human pathogen and its isolation from meat products indicate the need for knowledge of its survival characteristics in meats. Thermal death times (D-values) for a single strain and a five-strain composite were determined in 1% peptone and autoclaved ground chicken meat at temperatures ranging from 49 to 57 degrees C. Survival was determined for these strains in chicken meat at 4, 23, 37, and 43 degrees C. Survival was also determined on raw chicken drumsticks stored at 4 degrees C in either an ambient or a CO2 atmosphere. D-values were greater in chicken meat than in peptone in all cases. D-values in peptone for strain H-840 at 49, 51, 53, 55, and 57 degrees C were 15.2, 4.90, 1.71, 0,64, and 0.25 min, respectively. The corresponding D-values in ground chicken meat were 20.5, 8.77, 4.85, 2.12, and 0.79 min, respectively. Similar results were obtained with a composite of five strains. When sterile ground chicken meat was inoculated with approximately 10(6) to 10(7) C. jejuni cells per g and stored at 37 degrees C in an ambient atmosphere, a 1-to 2-log count increase occurred during the first 4 days, followed by a gradual decline of about 1 log during the remainder of the 17-day storage period. No growth was observed among similarly inoculated samples that were stored at 4, 23, and 43 degrees C but counts declined by about 1 to 2 logs at 4 degrees C (17 day), by 2.5 to 5 logs at 23 degrees C (17 days), and to undetectable levels at 43 degrees C (between 10 and 16 days). Survival on raw chicken drumsticks stored at 4 degrees C in CO2 and in an ambient atmosphere declined by about 1.5 and 2.0 logs, respectively, during 21 days of storage. The effect of temperature on the survival of C. jejuni in chicken meat was similar to that reported in other natural and laboratory milieus. Ordinary cooking procedures that destroy salmonellae would be expected to destroy C. jejuni.     

Specific detection and confirmation of Campylobacter jejuni by DNA hybridization and PCR.

Conventional detection and confirmation methods for Campylobacter jejuni are lengthy and tedious. A rapid hybridization protocol in which a 1,475-bp chromogen-labelled DNA probe (pDT1720) and Campylobacter strains filtered and grown on 0.22-micron-pore-size hydrophobic grid membrane filters (HGMFs) are used was developed. Among the environmental and clinical isolates of C. jejuni, Campylobacter coli, Campylobacter jejuni subsp. doylei, Campylobacter lari, and Arcobacter nitrofigilis and a panel of 310 unrelated bacterial strains tested, only C. jejuni and C. jejuni subsp. doylei isolates hybridized with the probe under stringent conditions. The specificity of the probe was confirmed when the protocol was applied to spiked skim milk and chicken rinse samples. Based on the nucleotide sequence of pDT1720, a pair of oligonucleotide primers was designed for PCR amplification of DNA from Campylobacter spp. and other food pathogens grown overnight in selective Mueller-Hinton broth with cefoperazone and growth supplements. All C. jejuni strains tested, including DNase-producing strains and C. jejuni subsp. doylei, produced a specific 402-bp amplicon, as confirmed by restriction and Southern blot analysis. The detection range of the assay was as low as 3 CFU per PCR to as high as 10(5) CFU per PCR for pure cultures. Overnight enrichment of chicken rinse samples spiked initially with as little as approximately 10 CFU/ml produced amplicons after the PCR. No amplicon was detected with any of the other bacterial strains tested or from the chicken background microflora. Since C. jejuni is responsible for 99% of Campylobacter contamination in poultry, PCR and HGMF hybridization were performed on naturally contaminated chicken rinse samples, and the results were compared with the results of conventional cultural isolation on Preston agar. All samples confirmed to be culture positive for C. jejuni were also identified by DNA hybridization and PCR amplification, thus confirming that these DNA-based technologies are suitable alternatives to time-consuming conventional detection methods. DNA hybridization, besides being sensitive, also has the potential to be used in direct enumeration of C. jejuni organisms in chicken samples.